Project Summary/Abstract Adolescent Idiopathic Scoliosis (AIS) is a prevalent condition that impacts 3% of children worldwide. There is currently no known underlying basis for the three-dimensional spinal curves that occur in AIS and, as such, treatment is restricted to invasive surgical intervention or bracing post onset. A major obstacle to current understanding and treatment of AIS is our poor knowledge of the underlying etiology of the condition, a situation which is further compounded by our lack of animal models. The objective of this proposal is to address these barriers to progress by elucidating the cause of AIS, by generating and characterizing exceptional zebrafish models of the condition, and by understanding the mechanistic basis of the disease. In my preliminary data, I demonstrate that zebrafish mutants with abnormal cilia motility and CSF flow exhibit late-onset spinal curves that model the defining features of human AIS. Moreover, I demonstrate that the cause of spinal curves in zebrafish ptk7 mutants, the only existing AIS model, is likely to originate with dysfunctional cilia. This led me to propose a novel hypothesis for the cause of AIS: abnormal cerebrospinal fluid (CSF) flow, which is generated in part by cilia, causes the abnormal spinal curves in AIS. To test this central hypothesis, I will examine by localization and proteomic studies the function of C21ORF59, a protein I have found to be critical for cilia-mediated flow generation by linking cilia motility and cilia polarity. Since the links between these two facets of cilia biology, both critical for productive flow generation, are poorly understood, my novel proteomic approaches will lead to an important increase in knowledge. As well as investigating the molecular basis of flow generation by cilia, I will generate and characterize several zebrafish AIS models to assess whether these have defective CSF flow and to test whether mutations in genes linked to human AIS affect CSF flow generation in zebrafish. This will remove the major barrier to progress in this field. I will also investigate whether human variants in cilia motility genes cause AIS in animal models. These experiments will allow me to decipher how abnormal cilia motility and dysfunctional CSF flow are linked to human AIS. Lastly, beginning in the mentored phase but extending into the independent phase, I will perform mechanistic experiments to define the spatial and temporal basis for AIS onset in zebrafish, experiments that require the use of our unique temperature sensitive cilia motility mutant. This will allow me to begin to explain how dysfunctional CSF flow causes spinal curves. My proposal will test whether spinal curves can be resolved post-onset by restoring CSF flow and the extent of that recovery, potentially providing a proof-of-principle that spinal curves can be treated non-invasively and paving the way for future therapeutic interventions for this prevalent and poorly understood disease. Since my work strongly suggests that CSF flow is critical to maintain spine straightness, I end with a series of targeted experiments aimed at understanding how CSF flow is sensed in the developing spinal canal.